Oropharyngeal candidiasis, or thrush, is a frequent disease among immunocompromised patients as well as in elderly people and young children. Overgrowth of Candida albicans in the oral cavity is caused by decreases in saliva or reduction in mucosal antifungal peptides such as human -defensins (hBDs). Histatins are a family of histidine-rich cationic proteins secreted by major salivary glands that contribute significantly to the antifungal activity of saliva. Histatin 5 (Hst 5) has the highest fungicidal activity of the family, and kills yeast by causing selective release of intracellular ions and nucleotides from C. albicans. HBDs share some similarities in fungicidal pathways with Hst 5, but their precise mechanism of toxicity is not known. Cytotoxicity of Hst 5 is initiated by binding to the cell wall followed by cytosolic transport where it disrupts intracellular ion tranport and causes hyperosmotic stress. We have identified cell wall localized C. albicans Ssa2 proteins that are chaperone proteins assisting intracelluar transport, but the molecular identity of the transport mechanism is not known. Our preliminary data show that permeases and endocytosis are involved in peptide translocation. A significant barrier to delivery of Hst 5 or other related cationic peptides is disruption of initial binding of Hst 5 to the yeast cell wall surface by extracellular salts. Objectives of this proposal are design of salt-insensitive peptides which are efficiently transported into the cell as a basis for development of peptide- based therapeutic agents for candidiasis. Therefore, our aims are to identify minimal domains of Hst 5 that function under high salt conditions, enhance their salt resistance with helix-capping motifs, and verify that they retain optimal transport properties. Microarray analyses of Hst 5 treated C. albicans cells show that osmotic stress response by Hog1 MAPKinase pathway is an important mechanism for recovery of cells from Hst 5 toxicity. Candidal cells exposed to physiological levels of antifungal peptides in the oral environment may develop resistance through activation of Hog1p. Our overall hypothesis is that defining stress response pathways and the sensors that initiate signaling will guide strategies to overcome Candidal adaptive resistance. The objectives of the proposed studies are to identify key elements required for fungal cell uptake of peptides and subsequent adaptive responses that modulate Hst 5 and hBD toxicity. This approach will support our long-range goal to develop alternative peptide- based therapies for treatment of oral candidiasis, which is currently limited to a small group of antifungal drugs.